Methods and systems for cell differentiation using optogenetics

Abstract

Methods and systems for differentiating cells in a cell population into a desired cell lineage are provided herein. In some embodiments, the methods and systems use light (e.g., optogenetics) to control the differentiation of cells into a desired cell lineage. Optionally, the methods and systems include differentiating the cells in suspension culture for large-scale production of cells of the desired cell lineage.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 347,780, filed on June 1, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Transcription factors can regulate the differentiation of (e.g., stem) cells into different cell types. Expression of a transcription factor such as MyoD can lead to cells that differentiate into muscle cells, while expression of other transcription factors such as PPARγ can cause differentiation of cells into adipocytes or adipocyte-like cells. Differentiation of a (e.g., stem) cell population into a single cell lineage, e.g., with high efficiency and temporal precision, can be difficult.

Summary of the Invention

[0003] The methods and systems described herein satisfy an unmet need for efficient differentiation of a cell (e.g., stem cell) population into a specific or desired cell lineage (or cell type), e.g., using optogenetics.

[0004] In one aspect, a method is provided for differentiating at least one cell of a cell population into a desired cell lineage, the method comprising: (a) providing or obtaining a cell population in a suspension culture; and (b) controlling the differentiation of at least one cell of the cell population with light, thereby differentiating at least one cell into a desired cell lineage. Optionally, the step of controlling the differentiation in (b) comprises irradiating at least one cell with light of a first wavelength. Optionally, the step of controlling the differentiation in (b) comprises removing light of a first wavelength from at least one cell. Optionally, at least one cell is genetically engineered to contain an exogenous nucleic acid encoding at least one transcription factor, at least one differentiation factor, or both, which brings about differentiation into a desired cell lineage. Optionally, the expression of at least one transcription factor, at least one differentiation factor, or both is induced by irradiating or removing light. Optionally, the exogenous nucleic acid comprises at least one promoter operably linked to at least one transcription factor, at least one differentiation factor, or both. Optionally, at least one cell is genetically engineered to contain an exogenous nucleic acid encoding at least one light-controllable transcriptional regulator. Optionally, the promoter is an inducible promoter. Optionally, at least one light-controllable transcriptional regulator is a light-controllable transcriptional activator. Optionally, the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain. Optionally, irradiating induces the light-controllable transcriptional activator to bind to and activate the inducible promoter, thereby causing the expression of at least one transcription factor, at least one differentiation factor, or both. Optionally, removing induces the light-controllable transcriptional activator to bind to and activate the inducible promoter, thereby causing the expression of at least one transcription factor, at least one differentiation factor, or both. Optionally, the promoter is a constitutive promoter. Optionally, at least one light-controllable transcriptional regulator is a light-controllable transcriptional repressor.In some cases, the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain. In some cases, irradiation induces the light-controllable transcriptional repressor to dissociate from the constitutive promoter, thereby causing the expression of at least one transcription factor, at least one differentiation factor, or both. In some cases, removing light induces the light-controllable transcriptional repressor to dissociate from the constitutive promoter, thereby causing the expression of at least one transcription factor, at least one differentiation factor, or both. In some cases, the exogenous nucleic acid further comprises a blocking sequence downstream of at least one promoter, and the blocking sequence, when present, blocks the expression of at least one first transcription factor, at least one differentiation factor, or both. In some cases, at least one cell further comprises a nucleic acid sequence encoding at least one light-controllable recombinase. In some cases, at least one light-controllable recombinase is a photoactivatable recombinase. In some cases, the photoactivatable recombinase comprises a recombinase or a portion thereof fused to a photoactivatable domain. In some cases, the blocking sequence is adjacent to a recombinase recognition site recognized by at least one light-controllable recombinase. In some cases, irradiation activates the light-controllable recombinase, thereby resulting in excision of the blocking sequence and inducing the expression of at least one transcription factor, at least one differentiation factor, or both. In some cases, at least one cell is a stem cell. In some cases, the stem cell is a pluripotent stem cell or a multipotent stem cell. In some cases, at least one cell is a fibroblast. In some cases, at least one cell is a human cell, a bovine cell, or a mouse cell. In some cases, the desired cell lineage is selected from the group consisting of adipocytes, myocytes, and chondrocytes. In some cases, at least one transcription factor is selected from the group consisting of PPAR gamma, CEBP alpha, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, and any combination thereof.In some cases, irradiating further includes irradiating light onto a plurality of cells in a cell population to differentiate each of the plurality of cells into a desired cell lineage, or removing further includes removing light from a cell population to differentiate each of the plurality of cells into a desired cell lineage. In some cases, the suspension culture has a volume of at least 100 milliliters (mL). In some cases, the suspension culture is contained within a bioreactor vessel. In some cases, the bioreactor vessel has an overall volume of at least 100 milliliters (mL). In some cases, the cell population grows on the surface of microcarriers. In some cases, the microcarriers are coated with one or more extracellular matrix components.

[0005] In another aspect, a system for differentiating a cell population is provided, the system comprising: (a) a cell population in a suspension culture medium, wherein at least one cell of the cell population is engineered to contain an exogenous nucleic acid encoding at least one transcription factor, at least one differentiation factor, or both, that bring about differentiation into a desired cell lineage, and the expression of the at least one transcription factor, the at least one differentiation factor, or both, is controlled by light; and (b) one or more light sources configured to irradiate at least one cell of the cell population with light of a first wavelength. Optionally, the exogenous nucleic acid comprises at least one promoter operably linked to the at least one transcription factor, the at least one differentiation factor, or both. Optionally, at least one cell is genetically engineered to contain an exogenous nucleic acid encoding at least one light-controllable transcriptional regulator. Optionally, the promoter is an inducible promoter. Optionally, the at least one light-controllable transcriptional regulator is a light-controllable transcriptional activator. Optionally, the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain. Optionally, upon irradiation with light of the first wavelength, the light-controllable transcriptional activator binds to and activates the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. Optionally, upon removal of the light of the first wavelength, the light-controllable transcriptional activator binds to and activates the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. Optionally, the promoter is a constitutive promoter. Optionally, the at least one light-controllable transcriptional regulator is a light-controllable transcriptional repressor. Optionally, the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain. Optionally, upon irradiation with light of the first wavelength, the light-controllable transcriptional repressor dissociates from the constitutive promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both.In some cases, the light-controllable transcriptional repressor dissociates from the constitutive promoter upon removal of light of the first wavelength, thereby causing the expression of at least one transcription factor, at least one differentiation factor, or both. In some cases, the exogenous nucleic acid further comprises a blocking sequence downstream of at least one promoter, and the blocking sequence, if present, blocks the expression of at least one transcription factor, at least one differentiation factor, or both. In some cases, at least one cell of the cell population further comprises a nucleic acid sequence encoding at least one light-controllable recombinase. In some cases, the at least one light-controllable recombinase is a photoactivatable recombinase. In some cases, the photoactivatable recombinase comprises a recombinase fused to a photoactivatable domain. In some cases, the blocking sequence is adjacent to a recombinase recognition site recognized by at least one light-controllable recombinase. In some cases, the blocking sequence is excised by at least one light-controllable recombinase, thereby being configured to induce the expression of at least one transcription factor. In some cases, the at least one cell is a stem cell. In some cases, the stem cell is a pluripotent stem cell or a multipotent stem cell. In some cases, the at least one cell is a fibroblast. In some cases, the at least one cell is a human cell, a bovine cell, or a murine cell. In some cases, the desired cell lineage is selected from the group consisting of adipocytes, myocytes, and chondrocytes. In some cases, the at least one transcription factor is selected from the group consisting of PPAR gamma, CEBP alpha, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX3, SOX9, SOX5, SOX6, and any combination thereof. In some cases, the system is a plurality of microcarriers, and the cell population further comprises a plurality of microcarriers that grow on the surface of the plurality of microcarriers. In some cases, the plurality of microcarriers are coated with one or more extracellular matrix components. In some cases, the one or more light sources comprise one or more light-emitting diodes (LEDs).In some cases, one or more light sources include one or more lasers. In some cases, one or more light sources include incandescent light sources.

[0006] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

[0007] The novel features of the present disclosure are described in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which describes exemplary embodiments in which the principles of the present disclosure are utilized and the accompanying drawings.

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DETAILED DESCRIPTION OF THE INVENTION

[0008] The developmental fate of a cell (e.g., a stem cell) can be determined by expressing a specific transcription factor in a cell that operates a developmental program leading to its differentiation into a specific cell type. However, control (e.g., temporal control) over the differentiation of a population of cells (e.g., stem cells) into one or more specific or desired cell lineages or cell types in a controlled manner can be difficult using existing techniques. For example, the differentiation of stem cells into a specific or desired cell type (e.g., the specific or desired cell type includes all or part of a three-dimensional tissue) may require high temporal or spatio-temporal precision. As described herein, control of the (e.g., temporal) expression of transcription factors in stem cells can enable the (e.g., simultaneous and / or patterned) differentiation of a single population of stem cells into a population of cells including one or more cell types. For example, the methods and systems disclosed herein can be used to differentiate a single population of (e.g., stem) cells into a single specific or desired cell type while minimizing or preventing the differentiation of all or part of the single population of cells into cell types other than the specific or desired cell type. The methods and systems provided herein generally use optogenetics to control the differentiation of cells (e.g., stem cells) into a desired cell lineage. In some examples, the methods and systems provided herein control the expression of transcription factors and / or differentiation factors that result in the differentiation of (e.g., stem) cells into a desired cell lineage using light (e.g., by irradiating the (e.g., stem) cells with light of a specific wavelength or wavelength range or by removing light of a specific wavelength or wavelength range from the (e.g., stem) cells), using a light-controllable transcriptional regulator (e.g., a light-controllable transcriptional activator, a light-controllable transcriptional repressor, etc.). In other examples, the methods and systems disclosed herein use a light-controllable recombinase (e.g., a photoactivatable recombinase) that is controlled by light to differentiate (e.g., stem) cells into a desired cell lineage.

[0009] Generally, the methods and systems provided herein use optogenetics to activate a desired differentiation pathway in cells (e.g., stem cells). The use of optogenetics to differentiate cells is relatively inexpensive compared to conventional methods (e.g., methods that use expensive chemical factors). Further, by not requiring the addition of exogenous chemical factors, the methods and systems provided herein are a safe alternative to conventional methods, particularly when differentiated cells are produced for downstream use by an individual (e.g., for consumption by an individual or for therapeutic use). In addition, the use of light to differentiate cells allows individual cells or subsets of a population of cells, rather than the entire population of cells, to be irradiated with light, as is typical with the addition of chemical factors. This provides precise temporal and spatial control of differentiation in a cell population. Further, in some embodiments, the methods and systems provided herein are adjustable because light can be easily supplied or removed to induce or block the expression of a differentiation pathway (e.g., when a light-inducible promoter is used). Also, the amount of gene expression can be controlled, for example, by adjusting the intensity of the light.

[0010] Advantageously, the methods and systems provided herein can differentiate cells (e.g., stem cells) in suspension culture into a desired cell lineage. The use of suspension culture enables the large-scale production of the desired cell type, which cannot be achieved with cells in adherent culture (e.g., placed in the wells of a tissue culture plate). Thus, the methods and systems provided herein are particularly advantageous when the large-scale production of a particular type or lineage of cells is desired.

[0011] In some embodiments, the methods and systems provided herein use optogenetics to control (e.g., temporally and / or spatially) the differentiation of cells into a desired cell lineage or cell type. For example, in some embodiments, a promoter (e.g., its expression) that regulates one or more transcription factors and / or differentiation factors (e.g., involved in a differentiation pathway) can be controlled by light. In some cases, the methods and systems can involve the use of a light-controllable transcriptional regulator that can bind to a promoter (or a region adjacent or proximal to the promoter) or dissociate from the promoter (or a region adjacent or proximal to the promoter) to activate the expression of one or more transcription factors and / or differentiation factors. In some cases, the light-controllable transcriptional regulator is a transcriptional regulator fused to a light-controllable domain (e.g., a light switch). In some cases, the light-controllable transcriptional regulator is a light-controllable transcriptional activator. The light-controllable transcriptional activator can be a transcriptional activator fused to a light-controllable domain (e.g., a light switch). In some cases, the light-controllable transcriptional regulator is a light-controllable transcriptional repressor. The light-controllable transcriptional repressor can be a transcriptional repressor fused to a light-controllable domain. Advantageously, such methods and systems do not result in permanent activation of a differentiation pathway, and the differentiation pathway can be switched on or off simply by supplying or removing light, as described herein.

[0012] In some embodiments, the methods and systems provided herein use one or more light-controllable recombinases. Recombinases recognize specific DNA sequences and, when two recognition sequences are in the appropriate arrangement, can excise or invert the orientation of DNA between the two sites. By activating the recombinase for a short time, a permanent change can be made to the DNA, which offers the prospect of permanently switching on a differentiation gene with only a short activation period. Such methods and systems can, for example, result in permanent activation of a differentiation program.

[0013] For example, methods and systems for differentiating a population of (e.g., stem) cells (e.g., individual (e.g., stem) cell populations) using suspension cell culture techniques are disclosed herein. In some cases, the systems described herein can be configured (and / or used) to differentiate a first population of (e.g., stem) cells into cells of a desired cell lineage (e.g., the cells of the desired cell lineage include one or more phenotypes, gene expression profiles, and / or epigenetic states that are different from the phenotype, gene expression profile, and / or epigenetic state of the undifferentiated cells). In some cases, the systems described herein can be used to perform the methods described herein. In some cases, the systems described herein can be useful for promoting the differentiation of a cell population into a specified or desired cell type, for example, by establishing and / or maintaining conditions necessary or beneficial for the differentiation of all or a portion of the cell population into the specified or desired cell type. In some cases, the systems described herein can be useful for promoting the differentiation of a cell population into a specified or desired cell type, for example, by establishing and / or maintaining conditions necessary to prevent the differentiation of all or a portion of the cell population into a cell type different from the specified or desired cell type (e.g., a cell type including additional phenotypes, gene expression profiles, and / or epigenetic states that are different from those of the specified or desired cell type). The methods and systems can, in some cases, include the use of light-controllable transcriptional regulators and / or light-controllable recombinases. A method for differentiating a (e.g., stem) cell population is provided herein. The method can include providing or obtaining a population of (e.g., stem) cells (e.g., engineered (e.g., stem) cells), and light-controlling the differentiation of at least one (e.g., stem) cell of the (e.g., stem) cell population into a desired cell lineage. Differentiation can, in some cases, include irradiating at least one (e.g., stem) cell of the (e.g., stem) cell population with light of a first wavelength to differentiate the at least one (e.g., stem) cell into the desired cell lineage.Differentiation may, in some cases, involve removing light of a specific wavelength (or completely removing light) to differentiate at least one (e.g., stem) cell into a desired cell lineage. In some examples, the (e.g., stem) cell population is in a suspension culture (e.g., in a bioreactor).

[0014] The expression of at least one transcription factor and / or differentiation factor in a cell can lead to the differentiation of the cell into a specific cell type. It is contemplated herein that any transcription factor or combination of transcription factors, and / or any differentiation factor or combination of differentiation factors, upon expression, can lead to the differentiation of the cell into a desired cell lineage. In some cases, the differentiation of a cell into a particular or desired cell lineage or cell type (e.g., adipocyte, myocyte, or chondrocyte) can be induced by a transcription factor, non-limiting examples of which include PPAR gamma, CEBP alpha, MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, PAX, SOX9, SOX5, SOX6, or any combination thereof. In a non-limiting example, when the differentiation of a cell into an adipocyte (e.g., fat cell) is desired, the cell can be induced to express, without limitation, transcription factors such as PPAR gamma and / or CEBP alpha. In another non-limiting example, when the differentiation of a cell into a myocyte (e.g., muscle cell) is desired, the cell can be induced to express, but not limited to, transcription factors such as MYOD, MYOG, Myf5, MRF4, HEYL, KLF4, and / or PAX3. In another non-limiting example, when the differentiation of a cell into a chondrocyte (e.g., cartilage cell) is desired, the cell can be induced to express, but not limited to, transcription factors such as SOX9, SOX5, and / or SOX6. In some embodiments, the at least one transcription factor is selected from the group consisting of: PPAR gamma, CEBP alpha, MYOD, MYOG, and combinations thereof. In some cases, the at least one differentiation factor can include at least one chromatin remodeling factor. In some cases, the chromatin remodeling factor can be SMARCD3 and / or JMJD3. In some cases, the expression of the transcription factor and / or differentiation factor can be regulated, for example, by light.

[0015] In certain embodiments, the methods described herein can include expressing a transcription factor and / or a differentiation factor (e.g., a chromatin remodeling factor) (e.g., that differentiates a (e.g., stem) cell into a desired cell lineage) in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 (e.g., stem) cells.

[0016] In some embodiments, the cell populations described herein (e.g., engineered cell populations) can contain at least one exogenous nucleic acid. The exogenous nucleic acid can contain sequences encoding any of the transcription factors and / or other differentiation factors (e.g., chromatin remodeling factors) described herein. The sequences encoding the transcription factors and / or other differentiation factors can be operably linked to a promoter. The promoter sequence can be constitutively active. In alternative embodiments, the promoter sequence can be conditionally active. For example, a conditionally active promoter sequence can be regulatable or inducible, e.g., by light. In some embodiments, the cells described herein contain at least two exogenous nucleic acids. In some embodiments, the cells described herein contain at least three, at least four, at least five, or more exogenous nucleic acids. Each exogenous nucleic acid can contain at least one nucleic acid sequence encoding one or more transcription factors and / or differentiation factors. The nucleic acid sequences encoding the transcription factors and / or differentiation factors can be operably linked to one or more promoters (e.g., constitutive, inducible) as described herein. Optionally, when multiple transcription factors and / or differentiation factors are used to differentiate the cells into a desired cell lineage, the expression of each of the multiple transcription factors and / or differentiation factors can be under the control of the same single promoter. In such cases, each gene encoding a transcription factor and / or differentiation factor can be combined in a single, bicistronic, or polycistronic transcript. The single transcript can encode a self-cleaving 2A peptide between the individual proteins, or can contain an internal ribosome entry site (IRES) within the sequence, or any combination thereof. In other cases, the expression of the transcription factors and / or differentiation factors can be under the control of different promoters.

[0017] In some cases, at least one (e.g., stem) cell of the (e.g., stem) cell population described herein can be genetically engineered to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one light-controllable transcriptional regulatory factor. In some cases, the at least one light-controllable transcriptional regulatory factor is a light-controllable transcriptional activator. A light-controllable transcriptional activator can be a transcriptional activator that is fused or otherwise associated with a light-controllable domain, such as a light switch (e.g., as described herein). In some cases, the at least one light-controllable transcriptional regulatory factor can be a light-controllable transcriptional repressor. A light-controllable transcriptional repressor can be a transcriptional repressor that is fused or otherwise associated with a light-controllable domain, such as a light switch (e.g., as described herein).

[0018] In various embodiments, the light-controllable transcriptional regulatory factor is a light-controllable transcriptional activator. In one embodiment, upon irradiation with light (e.g., at a particular wavelength or wavelength range), the light-controllable transcriptional activator binds to or otherwise associates with a promoter sequence (e.g., an inducible promoter), thereby inducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein).

[0019] Figures 1A, 1B, 2A, and 2B depict various embodiments for controlling the expression of one or more transcription factors and / or differentiation factors using a light-controllable activator. As shown in Figure 1A, in one embodiment, the transcriptional activator is fused to a first light heterodimerization domain (e.g., light heterodimerization domain 1). A second light heterodimerization domain (e.g., light heterodimerization domain 2) that can dimerize with the first light heterodimerization domain is fused to a DNA binding domain. The DNA binding domain binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second light heterodimerization domains do not associate, and the expression of the transcription factor and / or differentiation factor is turned off. Upon irradiation with light (e.g., at an appropriate wavelength or wavelength range), the first and second light heterodimerization domains heterodimerize, bringing the transcriptional activator into close proximity with an inducible promoter. The transcriptional activator then induces the activity of the inducible promoter, resulting in the expression of the transcription factor and / or differentiation factor and leading to differentiation into the desired cell lineage. In this scenario, cells irradiated with light differentiate into the desired cell lineage, while cells not irradiated with light or irradiated with light of an inappropriate wavelength remain undifferentiated.

[0020] In another embodiment, as shown in FIG. 1B, the transcriptional activator is fused to a photo-homodimerization domain and a DNA-binding domain. In this scenario, when the DNA-binding domain homodimerizes with another DNA-binding domain, it binds to a DNA sequence within or near the promoter sequence. Upon irradiation with light (e.g., light within an appropriate wavelength or wavelength range), the photo-homodimerization domain homodimerizes, thereby bringing the two DNA-binding domains into close contact so that they homodimerize. Upon homodimerization, the DNA-binding domain binds to the promoter sequence, bringing the transcriptional activator into close contact with the inducible promoter. The transcriptional activator then induces the activity of the inducible promoter, resulting in the expression of transcription factors and / or differentiation factors and leading to differentiation into the desired cell lineage. In this scenario, cells irradiated with light differentiate into the desired cell lineage, while cells not irradiated with light or not irradiated with light of the appropriate wavelength remain undifferentiated.

[0021] In various embodiments, the light-controllable transcriptional activator binds or associates with a promoter sequence (e.g., an inducible promoter) in the absence of light or in the absence of light of a specific wavelength, thereby inducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In the presence of light, the light-controllable transcriptional activator does not bind to or dissociate from the promoter sequence (e.g., an inducible promoter), thereby preventing or reducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein).

[0022] As shown in FIG. 2A, in one embodiment, the transcriptional activator is fused to a first photocleavable domain (e.g., photocleavable domain 1). A second photocleavable domain (e.g., photocleavable domain 2) is fused to the DNA binding domain. The DNA binding domain binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second photocleavable domains associate, bringing the transcriptional activator into close contact with the inducible promoter and turning on the expression of transcription factors and / or differentiation factors. When irradiated with light (e.g., within an appropriate wavelength or wavelength range), the first and second photocleavable domains dissociate, releasing the transcriptional activator from the inducible promoter, thereby turning off the expression of transcription factors and / or differentiation factors. In this scenario, cells irradiated with light remain undifferentiated, while cells not irradiated with light or irradiated with light of an inappropriate wavelength differentiate into the desired cell lineage.

[0023] In another embodiment, as shown in FIG. 2B, the transcriptional activator is fused to a photocleavable domain and a DNA binding domain. In this scenario, the DNA binding domain binds to a DNA sequence within or near the promoter sequence when it homodimerizes with another DNA binding domain. In the absence of light, the photocleavable domain homodimerizes with another photocleavable domain, allowing the DNA binding domain to homodimerize and bind to the inducible promoter, bringing the transcriptional activator into close contact with the inducible promoter. The transcriptional activator then induces the activity of the inducible promoter, resulting in the expression of transcription factors and / or differentiation factors and leading to differentiation into the desired cell lineage. When irradiated with light (e.g., within an appropriate wavelength or wavelength range), the photocleavable domain dissociates, releasing the transcriptional activator from the inducible promoter and turning off the expression of transcription factors and / or differentiation factors. In this scenario, cells irradiated with light remain undifferentiated, while cells not irradiated with light or irradiated with light of an inappropriate wavelength differentiate into the desired cell lineage.

[0024] In various embodiments, the light-controllable transcriptional regulator is a light-controllable transcriptional repressor. In one embodiment, the light-controllable transcriptional repressor, when irradiated with light (e.g., at a particular wavelength or wavelength range), binds to or otherwise associates with a promoter sequence (e.g., a constitutive promoter or a sequence between a constitutive promoter and a transcription start site), thereby repressing the constitutive promoter and preventing or reducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In the absence of light, the light-controllable transcriptional repressor does not bind to or dissociate from a promoter sequence (e.g., a constitutive promoter or a sequence between a constitutive promoter and a transcription start site), thereby allowing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In such cases, the (e.g., stem) cells irradiated with light remain undifferentiated, while the (e.g., stem) cells not irradiated with light differentiate into the desired cell lineage.

[0025] Figures 3A, 3B, 4A, and 4B depict various embodiments of controlling the expression of one or more transcription factors and / or differentiation factors using a light-controllable transcriptional repressor. As shown in Figure 3A, in one embodiment, the transcriptional repressor is fused to a first light heterodimerization domain (e.g., light heterodimerization domain 1). A second light heterodimerization domain (e.g., light heterodimerization domain 2) that can dimerize with the first light heterodimerization domain is fused to a DNA binding domain. The DNA binding domain binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second light heteromimetic domains do not associate, and the expression of the transcription factor and / or differentiation factor is turned on (e.g., because the gene encoding the transcription factor and / or differentiation factor is under the control of a constitutive promoter). When irradiated with light (e.g., at an appropriate wavelength or wavelength range), the first and second light heteromimetic domains dimerize, bringing the transcriptional repressor into close contact with the promoter sequence or the sequence between the promoter and the transcription start site. The transcriptional repressor blocks the activity of the constitutive promoter, and the expression of the transcription factor and / or differentiation factor is turned off. In this scenario, cells irradiated with light remain undifferentiated, while cells not irradiated with light or irradiated with light of an inappropriate wavelength differentiate into the desired cell lineage.

[0026] In another embodiment, as shown in Figure 3B, the transcriptional repressor is fused to a photo-homodimerization domain and a DNA-binding domain. In this scenario, when the DNA-binding domain homodimerizes with another DNA-binding domain, it binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second photo-homodimerization domains do not associate, and the expression of the transcription factor and / or differentiation factor is turned on (e.g., because the gene encoding the transcription factor and / or differentiation factor is under the control of a constitutive promoter). When irradiated with light (e.g., light within an appropriate wavelength or wavelength range), the first and second photo-homodimerization domains dimerize, bringing the transcriptional repressor into close contact with the promoter sequence or the sequence between the promoter and the transcription start site. The transcriptional repressor blocks the activity of the constitutive promoter, and the expression of the transcription factor and / or differentiation factor is turned off. In this scenario, cells irradiated with light remain undifferentiated, while cells not irradiated with light or irradiated with light of an inappropriate wavelength differentiate into the desired cell lineage.

[0027] In various embodiments, the light-controllable transcriptional repressor binds to or associates with a promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcription start site) in the absence of light, thereby preventing or reducing the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In the presence of light, the light-controllable transcriptional repressor does not bind to or dissociates from the promoter sequence (e.g., a constitutive promoter, or a sequence between a constitutive promoter and a transcription start site), thereby activating the expression of one or more downstream genes (e.g., one or more transcription factors and / or differentiation factors as described herein). In such cases, cells irradiated with light (e.g., stem cells) differentiate into the desired cell lineage, while cells not irradiated with light (e.g., stem cells) remain undifferentiated.

[0028] As shown in FIG. 4A, in one embodiment, the transcriptional repressor is fused to a first photorelease domain (e.g., photorelease domain 1). A second photorelease domain (e.g., photorelease domain 2) is fused to the DNA binding domain. The DNA binding domain binds to a DNA sequence within or near the promoter sequence. In the absence of light, the first and second photorelease domains associate, bringing the transcriptional repressor into close contact with the promoter sequence or the sequence between the promoter and the transcription start site, turning off the expression of transcription factors and / or differentiation factors. When irradiated with light (e.g., at an appropriate wavelength or wavelength range), the first and second photorelease domains dissociate, releasing the transcriptional repressor from the promoter sequence, thereby turning on the expression of transcription factors and / or differentiation factors. In this scenario, cells irradiated with light differentiate into the desired cell lineage, while cells not irradiated with light or not irradiated with light of the appropriate wavelength remain undifferentiated.

[0029] In another embodiment, as shown in FIG. 4B, the transcriptional repressor is fused to a photorelease domain and a DNA binding domain. In this scenario, the DNA binding domain binds to a DNA sequence within or near the promoter sequence when it homodimerizes with another DNA binding domain. In the absence of light, the photorelease domain homodimerizes with another photorelease domain, allowing the DNA binding domain to homodimerize and bind to the promoter sequence or the sequence between the promoter and the transcription start site, bringing the transcriptional repressor into close contact with the promoter sequence or the sequence between the promoter and the transcription start site. The transcriptional repressor blocks the activity of the constitutive promoter, and the expression of transcription factors and / or differentiation factors is turned off. When irradiated with light (e.g., at an appropriate wavelength or wavelength range), the photorelease domain dissociates, releasing the transcriptional repressor from the promoter or the sequence between the promoter and the transcription start site, and the expression of transcription factors and / or differentiation factors is initiated. In this scenario, cells irradiated with light differentiate into the desired cell lineage, while cells not irradiated with light or not irradiated with light of the appropriate wavelength remain undifferentiated.

[0030] Non-limiting examples of DNA-binding domains that can be used with any of these embodiments include Gal4 (which binds to the Gal4 UAS DNA sequence), TetR (which binds to the TetO DNA sequence), and CymR (which binds to the CuO DNA sequence).

[0031] In some cases, a transcription regulator (e.g., a transcriptional activator, a transcriptional repressor) is fused to a light-controllable domain. In some cases, the light-controllable domain is an optogenetic switch. In some cases, the optogenetic switch is a dimerization-based optogenetic switch. In some cases, the dimerization-based optogenetic switch is a heterodimerization-based optogenetic switch as described herein. In other cases, the dimerization-based optogenetic switch is a homodimerization-based optogenetic switch as described herein. Any light-controllable domain, including any of the light-controllable domains described herein, may be used. Non-limiting examples of optogenetic dimerization systems suitable for use with the methods and systems provided herein are described in Table 2.

[0032] Non-limiting examples of transcriptional activators include the transcriptional activation domain of VP16 of herpes simplex virus (HSV) and p65. Non-limiting examples of transcriptional repressors include the KRAB (Kruppel-associated box) domain of Kox1.

[0033] In other aspects of the disclosure, control of differentiation can involve the use of a light-controllable recombinase. In such a scenario, the expression of one or more transcription factors and / or differentiation factors can be under the control of a (e.g., constitutive) promoter. A blocking sequence can be inserted between the (e.g., constitutive) promoter and the sequence encoding one or more transcription factors and / or differentiation factors, such that when the blocking sequence is present, the expression of the one or more transcription factors and / or differentiation factors is blocked. When the blocking sequence is removed, the expression of the one or more transcription factors and / or differentiation factors is activated. Removal of the blocking sequence can be achieved by the use of a recombinase (e.g., a light-controllable recombinase). In such a scenario, the blocking sequence is adjacent to the recombinase recognition site, such that upon irradiation with light, the light-controllable (or light-activatable) recombinase excises the blocking sequence, thereby enabling the expression of the one or more transcription factors and / or differentiation factors. By fusing the recombinase to a light-controllable domain (e.g., a optogenetic switch (e.g., such as those described herein)), the recombinase can be activated by light (e.g., at a particular wavelength or wavelength range). In such a scenario, irradiation of the (e.g., stem) cells results in the differentiation of the (e.g., stem) cells into the desired cell lineage.

[0034] Figure 5 shows a non-limiting example of a nucleic acid cassette suitable for use with the disclosure herein. Promoter 503 can be operably linked to a nucleic acid sequence encoding transcription factor 504. Optionally, blocking sequence 502 can be positioned between the promoter sequence and the nucleic acid sequence encoding the transcription factor (e.g., the blocking sequence is downstream of the promoter). A blocking sequence can be any nucleic acid sequence that prevents transcription of the nucleic acid sequence encoding the transcription factor, e.g., a nucleic acid sequence encoding one or more stop codons and / or a transcription terminator sequence. Optionally, the blocking sequence can include an expression cassette or multiple expression cassettes, each containing a transcription terminator sequence. Optionally, the blocking sequence may be adjacent to a recombinase recognition site 501 (e.g., where the recombinase recognition site is recognized by a light-controllable recombinase). In such a scenario, in the presence of an activated recombinase, the blocking sequence is excised, allowing expression of the transcription factor. Optionally, the recombinase can be activatable, e.g., by light, as described herein, thus allowing control of recombinase activity. In such cases, the recombinase can be expressed in (e.g., stem) cells but can be in an inactive state until the (e.g., stem) cells are exposed to light of a particular wavelength or wavelength range. Exposure of the (e.g., stem) cells to light of a particular wavelength or wavelength range can activate the recombinase, resulting in excision of the blocking sequence and expression of the transcription factor (which in turn can lead to differentiation of the (e.g., stem) cells into a desired cell lineage).

[0035] In an alternative embodiment, instead of excision of the blocking sequence, an activatable recombinase can be used to invert a nucleic acid sequence such that the nucleic acid sequence is under the control of the promoter, thereby resulting in expression of the transcription factor and / or a differentiation factor.

[0036] In some cases, multiple recombinases can be used such that different transcriptional programs are activated depending on which recombinase is activated. For example, a cell can express multiple light-controllable recombinases (e.g., each fused to a different light-controllable domain). If differentiation into cell lineage A is desired, the cell is exposed to light of a first wavelength or wavelength range, thereby activating a first recombinase fused to a first light-controllable domain, thereby resulting in the expression of a first transcription factor and / or differentiation factor, and the differentiation of the cell into cell lineage A. If differentiation into cell lineage B is desired, the cell is exposed to light of a second different wavelength or wavelength range, thereby activating a second recombinase fused to a second different light-controllable domain, thereby resulting in the expression of a second transcription factor and / or differentiation factor, and the differentiation of the cell into cell lineage B. In a cell population, this method can be used to control (e.g., temporally) the differentiation of cells into a particular or desired cell lineage or cell type.

[0037] In some examples, the recombinase can be activated using a light-activatable system. The light-activatable system can be as described in Table 1.

[0038] [Table 1]

[0039] In various aspects, combinations of light-controllable domains (e.g., a first light-controllable domain and a second light-controllable domain) can be used (e.g., each of the light-controllable domains can be fused to a part of a recombinase). In some cases, the first light-controllable domain and the second light-controllable domain can be binding partners such that upon irradiation with light within a particular wavelength or particular spectral range, the first and second light-controllable domains heterodimerize or hetero-oligomerize. The first and second light-controllable domains heterodimerize or hetero-oligomerize upon irradiation with light within a particular wavelength or particular spectral range, thereby bringing protein domains (or functional domains or functional parts thereof) into close contact with each other, as a result of which the recombinase is activated.

[0040] In various aspects, the light-controllable domain includes a Light-Oxygen-Voltage (LOV) photoreceptor domain, an LOV2 photoreceptor domain, a cryptochrome (CRY) domain, a blue light-utilizing FAD (BLUF) photoreceptor domain, a phytochrome (PHY) domain, CIB1 (cryptochrome-interacting basic helix-loop-helix protein 1) (or a functional part or domain thereof, e.g., CIBN (the N-terminal domain of CIB1)), a PIF (phytochrome-interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin-binding domain (CBD), or a combination thereof.

[0041] In some examples, combinations of light-controllable domains are used, where the first light-controllable domain is cryptochrome 2 (or a variant or functional portion thereof), and the second light-controllable domain is CIB1 (or a variant or functional portion thereof, such as CIBN). In some examples, combinations of light-controllable domains are used, where the first light-controllable domain is BphP1 (or a variant or functional portion thereof), and the second light-controllable domain is QPAS1 (or a variant or functional portion thereof). Optionally, the light-controllable domain (or combination of photoactivatable domains) is selected from Table 2. Optionally, the light-controllable domain has an amino acid sequence having at least about 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity with any one of the light-controllable domains described in Table 2.

[0042]

Table 2

[0043] In another aspect of the methods described herein, the recombinase can be activated by a specific wavelength or wavelength range of light. The recombinase can be constitutively expressed in an inactive form. The recombinase can be conditionally expressed by a light-inducible system in an inactive form. The recombinase can mediate irreversible excision. The recombinase can be a serine integrase, such as ΦC31, TP901, and Bxbl. The recombinase can be a tyrosine recombinase, such as Cre, VCre, and Flp.

[0044] In another embodiment, dimerization and activation of the recombinase can be induced by light (e.g., optogenetic dimerization). The first half of the recombinase and the second half of the recombinase can be fused to a light-controllable domain. In various embodiments, the light-controllable domain can include a Light-Oxygen-Voltage (LOV) photoreceptor domain, an LOV2 photoreceptor domain, a cryptochrome (CRY) domain, a blue-light using FAD (BLUF) photoreceptor domain, a phytochrome (PHY) domain, CIB1 (cryptochrome interacting basic helix-loop-helix protein 1) (or a functional portion or domain thereof, e.g., CIBN), a PIF (phytochrome interacting factor) domain, a Dronpa domain, a UVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin binding domain (CBD), or a combination thereof. Regulation of dimerization and activation of the recombinase can utilize any of the light-controllable systems described in Table 2. Alternatively, dimerization and activation can be induced by temperature.

[0045] In various aspects, the method can include exposing cells (e.g., genetically engineered to express a fusion protein comprising a recombinase and a light-controllable domain) to light of a specific wavelength or light within a specific spectral range. The wavelength of the light can be selected such that the light can activate the light-controllable domain. For example, Table 2 provides non-limiting examples of light parameters for different light-controllable domain systems. The wavelength of the light can be one or more of infrared, near-infrared, visible light (e.g., red, green, blue), ultraviolet light, or combinations thereof. Infrared light can include light having wavelengths from about 780 nm to 1 mm. Near-infrared light can include light having wavelengths from about 740 nm to about 780 nm. Red light can include light having wavelengths of about 620 nm to 750 nm, 600 nm to 690 nm, or about 650 nm. Green light can include light having wavelengths from about 577 nm to about 492 nm. Blue light can include light having wavelengths of 492 to about 455 nm, or about 440 nm to about 473 nm. Ultraviolet light can include light having wavelengths from about 10 nm to 400 nm, or about 280 to 315 nm. In various aspects, the wavelength of the light is from 100 nm to 1 mm.

[0046] In some embodiments, the method and system include irradiating the cells with light of a specific intensity or intensity range. For example, the methods provided herein can include irradiating the cells with light having an intensity of about 2 pW / mm2. In other examples, the methods provided herein can include irradiating the cells with light having an intensity of about 8 pW / mm2. In some cases, the methods provided herein can include irradiating the cells with light having an intensity from about 2 pW / mm2 to about 8 pW / mm2. It should be understood that the level of light intensity can vary and can depend on the type of optogenetic switch used.

[0047] In some embodiments, the methods provided herein include irradiating the cells with light having an irradiation pattern, such as a pulse pattern. For example, the method can include illuminating the cells over a period of time, turning off the illumination of the cells over a period of time, and then repeating the on / off cycle a plurality of times.

[0048] In another embodiment, the recombinase comprises a single-chain polypeptide. The single-chain polypeptide can be fused to a light-controllable domain to create a light-controllable recombinase. Exposure to light of a specific wavelength can result in the activity of the recombinase. For example, irradiation of AsLOV2-based Cre LiCre with blue light results in the activation of the recombinase.

[0049] In another embodiment, the recombinase can be fused to a PhoCl protein or a derivative thereof. Irradiation with violet light (about 400 nm) results in the cleavage of PhoCl. In some examples, the PhoC1 domain can be present in a fusion protein between a blocker domain and a recombinase domain as shown in FIG. 2. The blocker domain can be any domain that prevents the recombinase from functioning, for example, a domain that prevents the recombinase from entering the nucleus. For example, the blocker domain can be a steroid receptor domain that interacts with Hsp90 to prevent nuclear entry of the recombinase. Exposure of a fusion protein containing a blocker domain, a PhoCl domain, and a recombinase to violet light cleaves the PhoCl domain, allowing the recombinase to enter the nucleus and reach genomic DNA, resulting in recombinase activity.

[0050] The recombinases, transcriptional regulators, transcription factors, differentiation factors, and other elements described herein can be encoded by nucleic acids. In some embodiments, the nucleic acids containing the recombinases, transcriptional regulators, transcription factors, differentiation factors, and other elements described herein can be expression cassettes or can be contained within an expression cassette. As used herein, "expression cassette" means a recombinant nucleic acid construct containing one or more nucleic acids described herein, where the recombinant nucleic acid construct is operably associated with at least one control sequence (e.g., a promoter).

[0051] In some cases, the engineered cells (e.g., the engineered cells described herein may be, for example, before or after differentiation) may contain a positive selection cassette or a negative selection cassette. For example, cells that are not a specific or desired cell lineage or cell type (e.g., undifferentiated cells or cells differentiated into a cell lineage or type other than the specific or desired cell lineage) can be selectively killed or eliminated from a cell population by applying one or more factors sufficient to induce each positive or negative selection cassette, including a negative selection cassette (e.g., in the nucleic acid sequence of a cell that is not excised due to recombinase activity) or a positive selection cassette (e.g., in the nucleic acid sequence of a cell that is excised due to recombinase activity). In some cases, by using a positive selection cassette in the nucleic acid sequence of a cell excised during recombinase activity (e.g., when sufficient stimulation to activate the positive selection cassette is provided), cells in which the recombinase activity was not successful can be removed or destroyed.

[0052] In certain embodiments, the nucleic acid is a component of a vector that can be used to introduce the nucleic acid into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a genomic integration vector or "integration vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector that can induce the expression of an operably linked gene is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like.

[0053] In a vector, regulatory elements such as promoters, enhancers, and polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral, or insect genes. A gene for the ability to replicate in a host (usually conferred by an origin of replication) and a selectable gene for facilitating recognition of transformants can be further incorporated. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, adeno-associated virus, etc. may be used. A plasmid vector can be linearized for integration into a chromosomal location. The vector may contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) to a defined location or a restricted set of sites in the genome. Further, the vector can contain sequences derived from transposable elements.

[0054] In some embodiments, the nucleic acid introduced into a eukaryotic cell is operably linked to a promoter and / or polyA signal, as known in the art. In some embodiments, a nucleic acid having a 5' end and a 3' end is operably linked to a promoter at the 5' end and a polyA signal at the 3' end. In some embodiments, the nucleic acid contains a 2A peptide sequence and / or an internal ribosome entry site.

[0055] In some embodiments, the expression cassette contains a nucleotide sequence encoding a selectable marker that can be used to select transformed host cells. As used herein, "selectable marker" means a nucleotide sequence that, when expressed, confers a different phenotype on a host cell expressing the marker and thus enables such transformed cells to be distinguished from cells that do not have the marker. Such nucleotide sequences can encode either a selectable marker or a screenable marker, depending on whether the marker confers a trait that can be selected by chemical means, such as using a selective agent (e.g., an antibiotic, etc.), or is merely a trait that can be identified by observation or testing, such as by screening (e.g., fluorescence).

[0056] Advantageously, the methods of the present specification provide differentiated (e.g., stem) cells in suspension (rather, e.g., two-dimensional adherent cell culture). In some cases, the methods provided herein do not require the use of a cell feeder layer. In some cases, the methods provided herein do not require the use of extracellular matrix components. In some cases, the method includes growing or maintaining cells (e.g., stem cells) in a bioreactor. In some cases, the methods provided herein may include the use of microcarriers (e.g., beads). In some cases, the microcarriers may be coated with an extracellular matrix component (e.g., promoting the attachment of (e.g., stem) cells thereto).

[0057] In various embodiments, the suspension culture has a volume of at least about 100 milliliters (mL). For example, the suspension culture may have a volume of at least about 150 mL, at least about 200 mL, at least about 250 mL, at least about 300 mL, at least about 350 mL, at least about 400 mL, at least about 450 mL, at least about 500 mL, at least about 550 mL, at least about 600 mL, at least about 650 mL, at least about 700 mL, at least about 750 mL, at least about 800 mL, at least about 850 mL, at least about 900 mL, at least about 950 mL, at least about 1000 mL, at least about 2000 mL, at least about 3000 mL, at least about 4000 mL, or at least about 5000 mL. In some examples, the suspension culture has a volume of less than about 1000 mL. In some examples, the suspension culture has a volume of more than about 1000 mL.

[0058] Any suitable cell culture medium for differentiating cells (e.g., stem cells) can be used. In some embodiments, the cell culture medium comprises, in any suitable combination, isotonic saline, buffer, amino acids, serum or serum replacement, sugars (e.g., glucose), and other exogenously added factors. In some embodiments, the cell culture medium comprises DMEM, F12, aMEM, Hepatostim™, RPMI, or a combination thereof, either in the presence or absence of serum. Suitable sera include bovine serum, fetal bovine serum, equine serum, etc. In some embodiments, serum supplements are used.

[0059] In some cases, the methods and systems provided herein can be used to differentiate (e.g., stem) cells into a desired cell lineage. In some cases, the cells used in the methods and systems provided herein are stem cells. The stem cells used in the methods and systems provided herein can be any desired stem cells. In some examples, the stem cells are pluripotent stem cells. In some examples, the stem cells are pluripotent stem cells. In some cases, the stem cells are embryonic stem cells. In some cases, the stem cells are mesenchymal stem cells. In some cases, the stem cells are satellite cells or muscle stem cells. In some cases, the stem cells are adipose stem cells. In one embodiment, the stem cells described herein are mammalian stem cells. In some cases, the mammalian stem cells are selected from the group consisting of human stem cells, bovine (cow) cells, ovine (sheep) stem cells, and porcine (pig) stem cells. In some cases, the stem cells are avian stem cells, e.g., but not limited to, chicken stem cells. In some cases, the stem cells are fish stem cells, e.g., but not limited to, tuna stem cells or salmon stem cells. In some cases, the cells can be non-stem cells (such as fibroblasts) that can be differentiated into a desired cell lineage. Any cell that can differentiate (e.g., any differentiable cell) can be used in the methods and systems provided herein. Any cell type including but not limited to human cells, bovine cells, or mouse cells can be used in the methods and systems provided herein.

[0060] In another aspect of the methods disclosed herein, a (e.g., stem) cell population is deposited on a solid support such as a microcarrier. Optionally, the (e.g., stem) cell population can grow on the surface of the microcarrier. Optionally, the microcarrier (or the portion thereof on which the cell population described herein grows) can be coated with one or more extracellular matrix components. The solid support may be biodegradable. The solid support may include natural materials. Examples of natural materials include, but are not limited to, extracellular matrix components, silk, gelatin, and alginate. The solid support may include synthetic materials. The solid support may include any surface or scaffold (e.g., hydrogel) to which (e.g., stem) cells can adhere.

[0061] The solid support can be coated with one or more extracellular matrix components. For example, the solid support can be coated with or incorporate collagen, hyaluronic acid, fibrin, fibronectin, integrin, laminin, proteoglycan, glycosaminoglycan, gelatin, vitronectin, or any other extracellular matrix protein.

[0062] Furthermore, a system (e.g., configured to implement the methods provided herein) is provided herein. In some cases, the system includes a population of (e.g., stem) cells in a suspension culture. In some cases, at least one (e.g., stem) cell of the population of (e.g., stem) cells is genetically engineered as described herein (e.g., to contain an exogenous nucleic acid comprising a nucleic acid sequence encoding at least one transcription factor and / or differentiation factor), resulting in differentiation into a desired cell lineage. In some cases, the expression of at least one transcription factor and / or differentiation factor is controllable by light (e.g., using a light-controllable transcriptional regulator and / or a light-controllable recombinase as described herein). In some embodiments, the system can further include one or more light sources configured to irradiate at least one (e.g., stem) cell of the population of (e.g., stem) cells with light of a specific wavelength or wavelength range. In some cases, the population of (e.g., stem) cells can be grown in a suspension culture in a bioreactor. In some cases, the one or more light sources are present on the bioreactor (e.g., on the surface of the wall of the bioreactor).

[0063] In some embodiments, the one or more light sources comprise one or more light-emitting diodes (LEDs). In some cases, the one or more LEDs include at least two different LEDs. In some cases, the at least two different LEDs emit light at different wavelengths. Additionally, or alternatively, the one or more light sources comprise one or more lasers. Additionally, or alternatively, the one or more light sources comprise an incandescent light source.

[0064] The bioreactor can be any type of culture vessel suitable for growing cells in a suspension culture. In various embodiments, the bioreactor vessel has a total volume of at least about 50 mL, at least about 100 mL, at least about 150 mL, at least about 200 mL, at least about 250 mL, at least about 300 mL, at least about 400 mL, at least about 500 mL, at least about 600 mL, at least about 700 mL, at least about 750 mL, at least about 800 mL, at least about 900 mL, at least about 1000 mL, at least about 2000 mL, at least about 3000 mL, at least about 5000 mL, or more.

[0065] The cells used in the systems provided herein can be any desired cells, including stem cells and non-stem cells (e.g., fibroblasts). In some examples, the stem cells are pluripotent stem cells. In some examples, the stem cells are pluripotent stem cells. In some cases, the stem cells are embryonic stem cells. In some cases, the stem cells are mesenchymal stem cells. In some cases, the stem cells are satellite cells or muscle stem cells. In some cases, the stem cells are adipose stem cells. In certain embodiments, the stem cells described herein are mammalian stem cells. In some cases, the mammalian stem cells are selected from the group consisting of human stem cells, bovine (cow) stem cells, ovine (sheep) stem cells, and porcine (pig) stem cells. In some cases, the stem cells are avian stem cells, e.g., but not limited to, chicken stem cells. In some cases, the stem cells are fish stem cells, e.g., but not limited to, tuna stem cells or salmon stem cells.

[0066] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or phrases used in this specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is generally understood in the art.

[0067] Throughout this application, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, a description of a range should be considered to specifically disclose all the possible sub-ranges within that range as well as the individual numerical values within that range. For example, a description of a range such as 1 - 6 should be considered to specifically disclose sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0068] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes plural samples, mixtures thereof.

[0069] As used in this specification, the term "about" refers to that number ± 10% of that number. The term "about" refers to the range obtained by subtracting 10% of the lowest value from that range and adding 10% of the highest value to that range.

[0070] Generally, "sequence identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining the "percent identity". The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. The percent identity can also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as discussed by Altschul et al., J. Mol. Biol. 215:403-410 (1990), Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly stated, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) and divides by the total number of symbols in the shorter of the two sequences. This program can be used to determine the percent identity over the full length of the protein being compared. Default parameters are provided, for example, for the blastp program to optimize searches with short query sequences. The program also allows for the use of the SEG filter to mask segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993).

[0071] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described.

Examples

[0072] Example 1: Recombinase-Mediated Excision to Activate Fate Cassette Expression A population of stem cells containing an exogenous nucleic acid sequence is cultured in suspension for differentiation. A pair of recognition sites recognized by a light-controllable recombinase is arranged in the construct such that when the corresponding recombinase is active in the cell, the muscle expression cassette (e.g., the fate determination cassette) is permanently turned on. In this scheme, one recognition sequence of the recombinase is arranged between the left-end (e.g., the most upstream) associated constitutive promoter and the transcription start site of the gene controlled by that promoter. The fate cassette is arranged at the opposite end of the construct. Before the fate cassette, there are the recognition sequence of the recombinase and the transcription start site, but no promoter. In this state, due to the absence of a promoter directly upstream to mobilize the transcription machinery due to the presence of the blocking sequence, the fate cassette is not expressed. When the cell is irradiated with light of a wavelength sufficient to activate the light-controllable recombinase and the recombinase becomes active in the cell, all the sequences between its two recognition sites are excised, and the fate cassette is carried immediately downstream of the right-end (the most downstream) recognition site under the control of the left-end (the most upstream) constitutive promoter. In this case, when the recombinase is activated, all the sequences between the promoter and the muscle expression cassette are excised, and the muscle expression cassette is expressed.

[0073] Example 2: Light-Induced Differentiation of Fibroblasts into Adipocyte-Like Cells in Suspension Culture This example shows the photoinduced differentiation of fibroblasts into adipocyte-like cells in suspension culture. In this example, a photoactivatable Cre recombinase was used to excise a blocking sequence and drive the expression of the adipocyte transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT / enhancer-binding protein α (C / EBPα).

[0074] Plasmid / Construct The plasmids created for this study were designed in Geneious (Biomatters). The different components of the plasmids were obtained either by restriction digestion, PCR, or DNA synthesis and assembled by Gibson cloning. Table 3 describes the plasmids used in these studies. pCMV-PPARγ, the Cre reporter plasmid, and the SV40 lentivirus were obtained from commercial sources.

[0075]

Table 3

[0076] Plasmid 703 (shown schematically in Figure 8B) has a constitutive human Ef1α promoter (P Ef1α) contains nucleic acid sequences encoding bovine transcription factor PPARg (UniProt: O18971-1) and fluorescent protein mScarletI (GenBank: APD76536.1) under the control of . The coding sequences of PPARg and mScarletI are linked by an IRES (Internal Ribosome Entry Site) sequence, enabling the co-expression of both genes. PPARg-IRES-mScarletI is linked to the promoter via a sequence containing three SV40 polyadenylation sequences (SV40pA) adjacent to loxP sites. Recombination of the two loxP sites via Cre recombinase excises the sequence containing the three SV40 polyadenylation sequences, turning on the expression of PPARg and mScarletI. Transcription from the Ef1α promoter terminates via the rabbit β-globin polyadenylation signal (rBGpA). The plasmid further contains an expression cassette for the neomycin resistance gene (Neo, GenBank: WP004614937.1) containing the constitutive human cytomegalovirus immediate early promoter (P CMV ) and the bovine growth hormone polyadenylation signal (bGHpA), which allows for the selection of cells with genomic integration of the described plasmid elements. The entire nucleotide sequence containing the described elements is flanked by the 5’ and 3’ ITR sequences of the PiggyBac transposon.

[0077] Plasmid 704 (schematically shown in Figure 7B) contains the constitutive human Ef1α promoter (P Ef1αIt contains nucleic acid sequences encoding bovine transcription factors PPARg (UniProt: O18971-1) and CEBPa (GenBank: NP_789741.2) and the fluorescent protein mScarletI (GenBank: APD76536.1). The coding sequences of PPARg and CEBPa are linked via a T2A self-cleaving peptide, and PPARg-T2A-CEBPa is linked to mScarletI by an IRES (internal ribosome entry site) sequence, enabling the simultaneous expression of the three genes. PPARg-T2A-CEBPa-IRES-mScarletI is linked to a promoter via a sequence containing three SV40 polyadenylation sequences (SV40pA) adjacent to loxP sites. Recombination of the two loxP sites via Cre recombinase excises the sequence containing the three SV40 polyadenylation sequences and turns on the expression of PPARg, CEBPa, and mScarletI. Transcription from the Ef1α promoter terminates via a rabbit β-globin polyadenylation signal (rBGpA). The plasmid further contains an expression cassette for the neomycin resistance gene (Neo, GenBank: WP004614937.1), which contains a constitutive human cytomegalovirus immediate early promoter (P CMV ) and a bovine growth hormone polyadenylation signal (bGHpA), which enables the selection of cells with genomic integration of the described plasmid elements. The entire nucleotide sequence containing the described elements is adjacent to the 5' and 3' ITR sequences of the PiggyBac transposon.

[0078] Plasmid 361 contains an expression cassette comprising a constitutive human cytomegalovirus immediate early promoter (P CMV ), the coding sequence of Cre recombinase (UniProt: P06956, residues 2-343) linked to a nuclear localization signal (UniProt: Q2HJ27, residues 320-328), and a bovine growth hormone polyadenylation signal (bGHpA).

[0079] Plasmid 709 (schematically shown in Figures 7A and 8A) contains a constitutive bovine Ef1α promoter (PEf1α ) It contains an expression cassette including the coding sequences of a light-inducible Cre recombinase and a fluorescent protein mTagBFP2 (GenBank: HCE4034942.1), as well as a bovine growth hormone polyadenylation signal (bGHpA). The coding sequences of the light-inducible Cre recombinase and mTagBFP2 are linked by an IRES (internal ribosome entry site) sequence, enabling the simultaneous expression of both genes. The light-inducible Cre recombinase contains three nuclear localization signals (UniProt: Q2HJ27, residues 320 - 328) fused to the N-terminus of an N-terminal truncated version of LiCre (described in Duplus-Bottin et al., “A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch”, eLife 10: e61268 (2021)), which includes AsLOV2 (Genbank: 4WF0_A, residues 11 - 148) and Cre (UniProt: P06956, residues 20 - 339). The entire nucleotide sequence containing the described elements is adjacent to the 5’ and 3’ IR / DR sequences of the Sleeping Beauty transposon.

[0080] P CMV - The PPARg lentiviral vector contains a nucleic acid sequence encoding the bovine transcription factor PPARg (UniProt: O18971-1) under the control of a constitutive human cytomegalovirus immediate early promoter (P CMV ). The lentiviral vector further contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a nucleic acid sequence encoding a puromycin resistance gene under the control of a constitutive mouse phosphoglycerate kinase 1 promoter (P mPGK ).

[0081] Cell line

[0082] Isolation of fibroblasts from Wagyu cattle

[0083] Adipose stem cells were extracted from fresh adipose tissue samples of adult Japanese Black beef bulls. Briefly, the adipose tissue was minced finely with a sterile scalpel and added to 25 ml of digestion solution (2 mg / ml collagenase II (Sigma, C2-BIOC), 1% penicillin / streptomycin / amphotericin B (Lonza, 17-745E), 10 μM ROCK inhibitor Y-27632 (Tocris, 1254)), made up to a total volume of 40 ml in a 50 ml centrifuge tube, and incubated at 37°C for 60 minutes with inversion every 2 minutes. The tube was centrifuged at 300 × g for 5 minutes to yield a pellet at the bottom and a fat plug at the top, which were discarded along with the supernatant. The pellet was washed with 25 ml of growth medium (DMEM (Sigma, SLM-021)) containing 10% fetal bovine serum (Avantor, 89510-186) and 1% penicillin / streptomycin / amphotericin B, then resuspended in 10 ml of growth medium and transferred to a T75 tissue culture flask. The cells were incubated at 37°C and 5% CO2 for 24 hours, after which the growth medium was changed every 2 days for multiple passages of subculture growth.

[0084] Generation of SV40 background cell line (SV40 cells)

[0085] Cells that stably express SV40-LgT (Large T antigen) were generated such that they confer a doubling time advantage and immortalize the cells. P3 Japanese Black fibroblast cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin / amphotericin B and maintained under standard conditions. Cells were transduced with lentivirus (Gentarget Inc LVP016-Hygro, San Diego, California, carrying LgT under the CMV promoter) in a 24-well plate, adding 20 μl of virus to each well and then incubating for 24 hours. After incubation, the medium was replaced with fresh culture medium. Approximately 48 hours later, the cells were subjected to antibiotic selection (hygromycin, InvivoGen, San Diego, California) for one week to promote the survival of only those cells stably transduced with the antibiotic resistance gene. These cells were then grown and, when they reached an appropriate number, several were cryopreserved for future use, including resuspension in cryopreservation medium, freezing at a controlled rate, and storage in liquid nitrogen.

[0086] Plasmid integration using PiyBac / Sleeping Beauty and selection using geneticin / cell sorting

[0087] One day before transfection, 500,000 SV40 cells were seeded into a T75 flask in growth medium. On the day of transfection, each transposon plasmid was co-transfected with the transposase plasmid (hyPB for PiggyBac and hySB100X for Sleeping Beauty) using Lipofectamine™ 3000 transfection reagent (ThermoFisher) according to the manufacturer's instructions. Each flask contained 9 μg of transposon plasmid, 0.9 μg of the corresponding transposase plasmid, 22 μL of P3000, and 27 μL of Lipofectamine™ 3000. Transfections without transposase were also performed to monitor the decrease in transient transfection signal prior to cell selection. The medium was changed 48 hours after transfection. For cells transfected with plasmid 703 and plasmid 704, 4 mg / ml of geneticin was included in the growth medium for selection. Control flasks containing the same number of unmanipulated cells were simultaneously selected with geneticin. All cells in the control flasks were killed on day 6, indicating that the surviving cells in the experimental flasks had been manipulated. The manipulated cells were grown in growth medium without geneticin for the experiment.

[0088] Select a cell population that stably expresses the plasmid

[0089] The cells were detached and dissociated into a single-cell suspension using TrypLE by incubating at 37 °C for 5 minutes. After centrifugation, the cells were resuspended in PBS and filtered through a 100 μm cell strainer. Cell sorting of the PiggyBac and Sleeping Beauty transposon integration cell populations was performed using a SONY cell sorter SH800S according to the manufacturer's instructions. To sort plasmid 709 in the 703 and 704 transfected cells, gating was performed on the mScarlet-negative BFP low and mScarlet-negative BFP high populations to purify Cre recombinase-expressing cells with an intact loxP-3XSTOP-1OXP sequence upstream of the transcription factor. All sorted cell populations were grown in growth medium.

[0090] Bioreactor preparation

[0091] A 100 ml spinner flask (Bellco) equipped with an impeller was coated with Sigmacote (Sigma Aldrich), excess Sigmacote was removed, and the flask was dried by removing the side cap and lid, inverting the flask, and placing them in the draft overnight. The next day, the flask was rinsed with 100 ml of fresh MilliQ water and dried. The dry reactor was assembled and autoclaved for 30 minutes in a high-speed / dry cycle.

[0092] Microcarrier preparation

[0093] A 50 ml Falcon tube was coated with Sigmacote, dried, washed with fresh MilliQ water, and dried upside down before use. 0.2 g of Cytodex 3 microcarriers were used per 100 ml flask. The microcarriers for each spinner flask were placed in a 50 mL Falcon tube coated with Sigmacote, and 45 mL of Mg / Ca 2+Hydration was carried out by adding PBS without [substance] to each Falcon tube. The Falcon tubes were inverted several times until the microcarriers were completely suspended and then left at room temperature for at least 3 hours. The supernatant was removed and the microcarriers were washed again with PBS without Mg / Ca 2+ After removing the supernatant, the microcarriers were washed again with PBS without Mg / Ca 2+ PBS without Mg / Ca was added up to the 40 ml mark and the microcarriers were sterilized by autoclaving for 15 minutes in a wet cycle. The rehydrated Cytodex 3 could be stored at 4 °C or used immediately. To use the microcarriers, 25 ml of the PBS supernatant was removed and 25 ml of growth medium containing DMEM / F12 high glucose, 10% FBS, 1% penicillin / streptomycin / amphotericin B was added. The microcarriers were allowed to settle and the medium wash was repeated twice. For a 100 mL container, the volume was made up to 20 mL.

[0094] Suspension culture

[0095] The microcarrier solution (20 ml) was inverted and poured into each spinner flask. The Falcon tubes were rinsed with 20 ml of growth medium containing DMEM / F12 high glucose + 10% FBS + 1% penicillin / streptomycin / amphotericin B and this was added to the flask. The spinner flask was then placed on a spinner plate and rotated at 30 rpm for at least 2 hours before the cells were added. The cells were washed, trypsinized and counted. 2 - 3 million cells were added to each reactor and the volume was made up to 100 ml with growth medium. The spinner flask was closed and swirled gently to mix the cells and microcarriers. The flask was then placed in an incubator and rotated at 30 rpm for 1 minute every hour for 3 hours and then left attached to the microcarriers overnight without rotation.

[0096] The next day, the spinner flasks were continuously placed on the spinner at 30 rpm. For each experiment, one flask was exposed to light and another flask was used as a control and not exposed to any light. The blue light (peak intensity at approximately 465 nm) used to irradiate the flasks was administered in 20 - second pulses every 80 seconds at an approximate output density of 8 μW / mm 2 on the reactor wall. To prevent light leakage into the dark control flasks, all flasks were covered with a light - shielding sheath and base together with aluminum foil. The irradiated flasks were exposed to the irradiation pattern for 48 hours. Two days after the suspension culture, the growth medium was replaced with StemPro adipogenic differentiation kit medium (Gibco) supplemented with 1% penicillin / streptomycin / amphotericin B. Then, 70% of the StemPro medium was replaced on day 3. To do this, the spinner flasks were removed from the spinner plate, the microcarriers were allowed to sediment for 5 minutes, and then 70 ml of warmed StemPro medium was exchanged. 1 mL of microcarrier / media was sampled per well for imaging, and this was done 3 times for each sample. When indicated, 2 mL of microcarrier / media was sampled twice for RNA. The cultures were sampled 6 days later in StemPro medium.

[0097] Staining

[0098] To stain with BODIPY 493 / 503, the wells were supplemented with PBS, most of the supernatant was removed without exposing the cells to air, and 1 mL / well of PBS (24 - well plate) containing 260 ng / mL of BODIPY 493 / 503 was gently added to the wells. The plate was incubated in a cell culture incubator at 37 °C, 5% CO2, and approximately 95% humidity for 30 minutes. 1 mL of the supernatant was gently removed from each well, and 1 mL of PBS was gently returned to each well for imaging.

[0099] To stain with Hoechst, 1 μg / ml of Hoechst in PBS was added to the samples for 30 minutes together with the BODIPY 493 / 503 staining.

[0100] Imaging

[0101] All 2D and 3D images were acquired using a Leica dmi8 THLJNDER microscope. More specifically, a 20x zoom with a 0.8 NA objective lens was used. To improve image quality and reduce noise, THUNDER’s Instant Computational Clearing (ICC) method was applied to all images during acquisition. The intensity, exposure time, and excitation / emission wavelengths of the Leica filter cubes for all fluorescent stains are listed below.

[0102] Hoechst: · Intensity: 31% · Exposure time: 100 ms · Excitation wavelength: 390 nm · Emission wavelength: 460 nm

[0103] BODIPY 493 / 503: · Intensity: 35% · Exposure time: 300 ms · Excitation wavelength: 510 nm · Emission wavelength: 535 nm

[0104] 2D Imaging

[0105] For all 2D assays, cells were imaged in a 96-well glass-bottom plate. On the 96-well plate, a 6x6 tile scan was performed. For all tile scans, the Leica AFC laser autofocus was used to ensure that the images were in focus across the wells.

[0106] 3D Imaging

[0107] Images were acquired on 6-well glass-bottom plates for assays comparing MBX, SV40, and SV40-PPARg cells in growth media and Stempro media (see below). All other 3D images were taken on 24-well glass-bottom plates. For all suspension experiments in both types of plates, two Z-stack images were manually taken at different positions within each well to ensure that the microcarriers were imaged. For each z-stack, dense positions with microcarriers were selected, and similar samples were taken across different wells and experiments.

[0108] Image analysis

[0109] For all image analyses, after post-processing with Leica as described below, the images were exported from Leica.lif files to individual.tif files using Python. Additionally, all plots were made using Python. Since BODIPY spots often aggregate together, the normalized BODIPY spot area occupied per image was chosen as the main metric across all suspension experiments. Thus, the occupied area was seen as a reliable measure of BODIPY staining and thus lipid droplet accumulation. For all analyses, any segmented BODIPY object with a diameter of 2 pixels or less was removed by filtering.

[0110] 2D image analysis

[0111] First, the Mosaic Merge function of the Leica software was used to stitch together the tile scans into a single image. The exported image was loaded into the CellProfiler pipeline. The RobustBackground segmentation algorithm of CellProfiler was used to segment BODIPY spots in 2D.

[0112] 3D image analysis

[0113] The Z-stack images were processed using the Leica's Max Projection function. This means that for every pixel in every image, the maximum intensity value of each channel across all z-planes was represented, resulting in a 2D image. These exported images were also loaded into CellProfiler. To segment the BODIPY staining on the microcarriers, the Otsu segmentation algorithm of CellProfiler was found to be optimal. Therefore, all the maximum projection images were segmented using the Otsu algorithm. The implementation of Otsu in CellProfiler was found to function better than the Leica's implementation for segmenting the BODIPY staining on the microcarriers. For data normalization, the microcarrier counts were done manually for each image.

[0114] qPCR

[0115] RNA was isolated from the collected cell samples using ZYMO Research’s Direct-zol RNA Miniprep kit and TRI reagent following strict RNase-free practices. The extracted RNA was then quantified and transcribed into cDNA via the Applied Biosystems™ High-Capacity RNA-to-cDNA™ kit. For qPCR, the cDNA was amplified using the Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix with appropriate primers. The prepared reaction mixture was loaded onto a Quantstudio 7Pro instrument. The amplification conditions were set as follows: UDG activation at 50 °C for 2 minutes, dual-lock DNA polymerase activation at 95 °C for 2 minutes, and 40 cycles of denaturation at 95 °C for 15 seconds and annealing / extension at 60 °C for 1 minute. The program was run in standard mode following the manufacturer's instructions. The bright-to-dark fold change was calculated by 2^-delta delta Ct.

[0116] Fibroblasts constitutively expressing PPARg that grow in adipogenic induction medium differentiate into adipocyte-like cells This example demonstrates that fibroblasts constitutively expressing PPARg, an adipogenic master regulator, differentiate into adipocytes in both two-dimensional culture and suspension culture when grown in adipogenic induction medium, but do not differentiate in adipogenic induction medium without constitutive PPARg expression.

[0117] In this example, wild-type bovine fibroblasts and fibroblasts transfected with SV40 large T antigen (SV40-LgT) (described above) were used. SV40-LgT gene expression confers a growth advantage and shortens the doubling time of fibroblasts. One day prior to transfection, 300,000 wild-type (WT) or SV40-expressing fibroblasts were seeded into 100 mm dishes in growth medium containing DMEM / F12 + 10% FBS + 1% penicillin / streptomycin / amphotericin B. The next day, after changing the growth medium, the CMV-PPARg lentiviral vector (Vector Builder) was added at an MOI of 10 for transfection, and polybrene was added at 8 μg / ml. After incubating the cells with the lentiviral vector for 48 hours, they were selected with 1.5 μg / ml puromycin in the growth medium. Control plates containing the same number of cells but not transfected with the viral vector were simultaneously selected with puromycin. All cells in the control plates were killed on day 6, indicating that the surviving cells in the transfection plates were transfected with the CMV-PPARg viral vector. For the experiment, the transfected cells were grown in growth medium containing DMEM / F12 + 10% FBS + 1% penicillin / streptomycin / amphotericin B without puromycin.

[0118] To demonstrate the differentiation of fibroblasts into adipocytes in two-dimensional culture, WT cells, WT cells transfected with CMV-PPARg, SV40 cells, and SV40 cells transfected with PPARg were first seeded at 3000 cells / cm in growth medium containing DMEM / F12 + 10% FBS + 1% penicillin / streptomycin / amphotericin B2 Seeding was carried out for 24 hours to enable cell attachment. The next day, the medium was replaced with StemPro adipogenic differentiation medium. After 3 days, the medium was changed by gently removing the supernatant without disturbing the oil droplets in the cells. The cells were cultured in the adipogenic medium for 6 days. Lipid staining was performed by staining with BODIPY 493 / 503 as described above. Imaging and image analysis were performed as described above.

[0119] As shown in FIGS. 9A and 9B, constitutive expression of PPARg in both WT cells (FIG. 9A, panel F) and SV40-expressing cells (FIG. 9A, panel H) resulted in enhanced lipid droplet formation (stained with BODIPY 493 / 503) in the presence of StemPro medium, an adipogenic induction medium, but did not result in enhanced lipid droplet formation in the presence of the growth medium (FIG. 9A, panels B and D). Adipogenic medium alone did not result in enhanced lipid droplet formation (FIG. 9A, panels E and G), demonstrating that PPARg expression in fibroblasts grown in adipogenic medium is required for lipid droplet accumulation and thus fibroblast differentiation into adipocyte-like cells in two-dimensional cell culture. FIG. 9B shows the quantification of FIG. 9A. As shown in FIG. 9B, when cells constitutively expressing PPARg are grown in the growth medium, lipid droplet accumulation is minimal. Similarly, when cells are grown in adipogenic medium without PPARg expression, lipid droplet accumulation is also minimal. In contrast, when cells constitutively expressing PPARg are grown in adipogenic medium, robust lipid droplet accumulation is present.

[0120] These results were further confirmed in three-dimensional suspension cultures. Spinner flasks were prepared as described above. Similar to the 3T3-L1 MBX cells, fibroblasts containing SV40 cells, with or without constitutive expression of PPARg (as described above), were cultured in a growth medium containing DMEM / F12 + 10% FBS + 1% penicillin / streptomycin / amphotericin B. Cells were seeded onto Cytodex 3 microcarriers as described above and cultured in suspension as described above, except that in some spinner flasks, the growth medium was switched to StemPro 1 day after seeding. The growth medium and StemPro were changed on day 3, and samples were harvested on day 6. The cell / microcarrier was stained according to the BODIPY 493 / 503 staining described above. 3T3-L1 MBX cells were used as a positive control. The 3T3-L1 MBX cells are a fibroblast cell line that has been induced to ensure nearly 100% differentiation into adipocyte-like cells when grown in an adipogenic medium.

[0121] As shown in Figure 10, 3T3-L1 MBX cells as well as SV40-CMV PPARg cells show accumulation of lipid droplets when grown in an adipogenic medium (Figure 10, panels D and F, respectively), but do not show accumulation of lipid droplets when grown in a growth medium (Figure 10, panels A and C, respectively). Non-transduced cells do not express adipocyte-like lipid droplets when grown in either a growth medium or an adipogenic medium (Figure 10, panels B and E, respectively), demonstrating that expression of PPARg+ adipogenic medium is required for the differentiation of fibroblasts into fat. This example confirms that fibroblasts are capable of differentiating into fat in suspension culture.

[0122] Verification of a photoactivatable recombinase construct

[0123] In this example, a plasmid encoding a photoactivatable recombinase was tested and verified for use in subsequent experiments. SV40 cells were seeded at a density of 6000 cells / well in a 96-well glass-like polymer bottom plate in growth medium the day before transfection. On the day of transfection, plasmid 709 (photoactivatable Cre recombinase) or plasmid 361 (wild-type Cre recombinase) was co-transfected with the Cre recombinase reporter plasmid (pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, Life Science Market, catalog number PVT11052) using Lipofectamine™ 3000 transfection reagent (ThermoFisher) according to the manufacturer's instructions. Each well contained 30 ng of Cre plasmid, 30 ng of Cre reporter plasmid, 0.12 μL of P3000, and 0.2 μL of Lipofectamine™ 3000. The Cre recombinase reporter plasmid expresses the fluorescent protein dsRed until Cre recombinase activity excises the coding sequence of dsRed located between two loxP sites and turns on the expression of the fluorescent protein eGFP. Since room light activates the recombinase and permanently changes the DNA, the cells were treated under red or green light. The medium in each well was changed 24 hours after transfection, and then the selected wells (2 μW / mm 2 or 8 μW / mm 2 of pulsed blue light (465 nm) (20 seconds on, 60 seconds off) were irradiated to start. Cells were harvested 48 hours after the start of irradiation for flow cytometry (Attune CytPix, Thermo Fisher Scientific) analysis. The recombination efficiency was calculated as follows. % recombination = % of EGFP-expressing cells / % of EGFP and % of mCherry-expressing cells, plotted as shown in Figure 11A, or, as shown in Figure 11B, the fold change in recombination in light vs. dark was calculated and plotted.

[0124] As shown in FIGS. 11A and 11B, in the absence of Cre recombinase, there was minimal leaky recombination. Constitutive expression of Cre (CMV-Cre) resulted in an efficiency of recombination of approximately 100% of the recombination that verified the reporter plasmid. The photoactivatable recombinase (plasmid 709) was verified and showed an increase in light output-dependent recombination with peak recombination at 8 μW / mm 2 of the two light intensities tested.

[0125] Differentiation of fibroblasts into adipocytes using a constitutively expressed recombinase that excises a blocking array and drives the expression of adipogenic transcription factors.

[0126] In this example, fibroblasts were differentiated into adipocytes in two-dimensional cell culture using constitutive Cre recombinase. This example verifies the plasmids used to differentiate fibroblasts into adipocytes.

[0127] 6000 cells / well stably expressing plasmid 703 or plasmid 704 (see Table 3) were seeded into 96-well glass-bottom polymer plates in growth medium (DMEM / F12 high glucose, containing 10% FBS, 1% penicillin / streptomycin / amphotericin B) the day before transfection. On the day of transfection, CMV-Cre plasmid (plasmid 361) was transfected using Lipofectamine™ 3000 transfection reagent (ThermoFisher) according to the manufacturer's instructions. Un-transfected controls were exposed to transfection reagent only. Each transfected well contained 60 ng of CMV-Cre, 0.12 μL of P3000, and 0.2 μL of Lipofectamine™ 3000. Forty-eight hours after transfection, the medium in some wells was changed to StemPro adipogenesis differentiation medium for 6 days. The growth medium and adipogenesis medium were changed after 3 days. BODIPY 493 / 503 staining of lipid droplets was performed on day 6 according to the procedure outlined above.

[0128] As shown in Figure 12, in cells transfected with plasmids 703 and 361 (Figure 12, panels B and F) or plasmids 704 and 361 (Figure 12, panels D and H), more lipid droplets were stained in each StemPro or proliferation medium experiment than in cells transfected with plasmid 703 (Figure 12, panels A and E) or 704 (Figure 12, panels C and G) alone. This demonstrated that the constitutively expressed Cre recombinase was able to excise the blocking sequences of plasmids 703 and 704 such that the expression of the transcription factors (PPARg for plasmid 703, or PPARg and CEBPa for plasmid 704) was turned on and differentiation into adipocyte-like cells occurred. Expression of PPARg alone and PPARg + CEBPa resulted in substantially more lipid droplet accumulation in StemPro adipogenic medium (Figure 12, panels F and H, respectively) than in proliferation medium (Figure 12, panels B and D), again emphasizing that both the transcription factors and the adipogenic medium were required to differentiate fibroblasts into fat. Figures 13A and 13B show the quantification of the results shown in Figure 12.

[0129] Differentiate fibroblasts into adipocytes using a photoactivatable recombinase to excise the blocking sequence and drive the expression of adipogenic transcription factors.

[0130] In this example, a photoactivatable Cre recombinase was used to differentiate fibroblasts into adipocytes in both two-dimensional cell culture and three-dimensional suspension culture.

[0131] In this experiment, fibroblasts expressing SV40-LgT (SV40 cells) were stably transfected with plasmid 704 (a construct enabling the expression of PPARg+CEBPa upon recombination) using the PiggyBac system. Cells were selected with antibiotics so that the entire population expressed plasmid 704. These cells were then stably transfected with plasmid 709 (a construct driving the constitutive expression of a photoactivatable recombinase) using the Sleeping Beauty system. Cells expressing plasmids 704 and 709 were sorted into populations with high and low expression of plasmid 709. Since room light activates the recombinase and permanently changes the DNA, the cells were treated under red or green light. The cells were then cultured in 2D and exposed to the following dark conditions, blue light (465 nm) with a pulse pattern of 20 s on, 60 s off at 2 μW / mm 2 or blue light (465 nm) with a pulse pattern of 20 s on, 60 s off at 8 μW / mm 2 . After 48 h of irradiation, the light was turned off and the medium was changed to adipogenic medium for 6 days. The cells were stained with BODIPY 493 / 503. As shown in Fig. 14, the increase in light power output induced an increase in lipid droplet accumulation in both cells with high expression of the photoactivatable recombinase (Fig. 14, panels A, B, and C) and cells with low expression of the photoactivatable recombinase (Fig. 14, panels D, E, and F). This data demonstrates that light induces the differentiation of fibroblasts into adipocytes in a power output-dependent manner. Figs. 15A and 15B show the quantification of the results presented in Fig. 14.

[0132] These results were further confirmed in three-dimensional suspension cultures. In this experiment, fibroblasts expressing SV40 LgT (SV40 cells) were stably transfected with plasmid 704 using the PiggyBac system (a construct that enables the expression of PPARg CEBPa upon recombination). Cells were selected with antibiotics such that the entire population expressed plasmid 704. These cells were then stably transfected with plasmid 709 (a construct driving the constitutive expression of a photoactivatable recombinase) using the Sleeping Beauty system. Cells expressing plasmids 704 and 709 were sorted into populations with high and low expression of plasmid 709. Since room light activates the recombinase and permanently changes the DNA, the cells were treated under red or green light. The cells were attached to Cytodex 3 microcarriers in 100 ml of growth medium and cultured in a spinner flask. During this time, the cells were kept in the dark or irradiated with blue light (465 nm) at a pulse pattern of 20 seconds on and 60 seconds off for 48 hours. After 48 hours, the illumination was turned off and the medium was replaced with 100 ml of StemPro adipogenic medium. The medium was changed after 3 days. The cells + microcarriers were then stained with Hoechst (which stains the nucleus) and BODIPY 493 / 503 (which stains lipid droplets). As shown in Figure 16, irradiation with blue light induced an increase in lipid droplet accumulation in both cells with high expression of the photoactivatable recombinase (Figure 16, panel H) and cells with low expression of the photoactivatable recombinase (Figure 16, panel F). This data demonstrates that light can be used to induce the differentiation of fibroblasts into adipocytes in suspension cultures. The images are z-projections, and representative images are shown in Figure 16. The area covered by BODIPY 493 / 503 staining normalized to the number of microcarriers in the image was quantified and is shown in Figure 17. 2 of blue light was irradiated in a pulse pattern of 20 seconds on and 60 seconds off for 48 hours. After 48 hours, the illumination was turned off and the medium was replaced with 100 ml of StemPro adipogenic medium. The medium was changed after 3 days. The cells + microcarriers were then stained with Hoechst (which stains the nucleus) and BODIPY 493 / 503 (which stains lipid droplets). As shown in Figure 16, irradiation with blue light induced an increase in lipid droplet accumulation in both cells with high expression of the photoactivatable recombinase (Figure 16, panel H) and cells with low expression of the photoactivatable recombinase (Figure 16, panel F). This data demonstrates that light can be used to induce the differentiation of fibroblasts into adipocytes in suspension cultures. The images are z-projections, and representative images are shown in Figure 16. The area covered by BODIPY 493 / 503 staining normalized to the number of microcarriers in the image was quantified and is shown in Figure 17.

[0133] As a control, SV40 cells (without expression of photoactivatable recombinase or adipogenic transcription factors) were attached to Cytodex 3 microcarriers in 100 ml of growth medium and cultured in a spinner flask. During this time, the cells were irradiated with blue light (465 nm) at 8 μW / mm 2 for 48 hours in a pulse pattern of 20 seconds on and 60 seconds off. After 48 hours, the illumination was turned off and the medium was replaced with 100 ml of StemPro adipogenic medium. The medium was replaced after 3 days. Then, the cells + microcarriers were stained with Hoechst (which stains nuclei) and BODIPY 493 / 503 (which stains lipid droplets). The images are z-projections. The area covered by BODIPY 493 / 503 staining normalized to the number of microcarriers in the image was quantified and shown in Figure 18. This data demonstrates that irradiation of SV40 cells with blue light alone in the absence of expression of photoactivatable recombinase and adipogenic transcription factors does not increase lipid droplet accumulation, and that expression of constructs encoding photoactivatable recombinase (at appropriate expression levels) and adipogenic transcription factors is required.

[0134] To demonstrate that genes related to adipogenesis were expressed in cells induced to differentiate into adipocyte-like cells using light, samples were taken from the cells shown in Figure 16, panels B and F. RNA was extracted and cDNA was generated. qPCR was performed to examine the expression of two important adipogenic genes, FABP4 and LPL. FABP4 encodes a fatty acid-binding protein found in adipocytes, and LPL encodes lipoprotein lipase expressed in adipose tissue. Figures 19A and 19B demonstrate that adipocyte-related genes are upregulated in the light compared to the dark, reflecting the increased lipid accumulation demonstrated in response to light as shown in Figure 16, panels B and F.

[0135] In summary, these experiments show that 8 μW / mm 2The blue light activated a photoactivatable recombinase that excised a blocking array, resulting in PPARg / CEBPa expression, induced lipid accumulation, and demonstrated that light differentiated fibroblasts into an adipocyte phenotype. They also demonstrated that the level of photoactivatable recombinase expression was important for the control of differentiation. Cells highly expressing the photoactivatable recombinase showed higher leaky photoactivatable recombinase activation in the dark, resulting in higher PPARg / CEBPa expression and lipid droplet accumulation in the dark compared to cells with low photoactivatable recombinase expression in the dark. This is thought to be because when the expression of the photoactivatable recombinase is too high, the possibility that the two halves of the photoactivatable recombinase come together increases to the point where the mechanism is less dependent on light. Therefore, having an appropriate level of photoactivatable recombinase expression is important for achieving the control of fibroblast differentiation into adipocyte-like cells in suspension using light.

[0136] Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present disclosure. It is to be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

**Claim 1** A method for differentiating at least one cell of a cell population into a desired cell lineage, comprising: (a) providing or obtaining a cell population in a suspension culture; and (b) controlling the differentiation of the at least one cell of the cell population with light, thereby differentiating the at least one cell into a desired cell lineage. A method comprising the above steps. **Claim 2** The method according to claim 1, wherein the step of controlling the differentiation in (b) comprises irradiating the at least one cell with light of a first wavelength. **Claim 3** The method according to claim 1, wherein the step of controlling the differentiation in (b) comprises removing light of a first wavelength from the at least one cell. **Claim 4** The method according to any one of claims 1 to 3, wherein the at least one cell is genetically engineered to contain an exogenous nucleic acid encoding at least one transcription factor, at least one differentiation factor, or both, which brings about differentiation into the desired cell lineage. **Claim 5** The method according to claim 4, wherein the expression of the at least one transcription factor, the at least one differentiation factor, or both is induced by irradiating or removing light. **Claim 6** The method according to claim 5, wherein the exogenous nucleic acid comprises at least one promoter operably linked to the at least one transcription factor, the at least one differentiation factor, or both. **Claim 7** The method according to claim 6, wherein the at least one cell is genetically engineered to contain an exogenous nucleic acid encoding at least one light-controllable transcriptional regulator. **Claim 8** The method according to claim 7, wherein the promoter is an inducible promoter. **Claim 9** The method according to claim 8, wherein the at least one light-controllable transcriptional regulator is a light-controllable transcriptional activator. **Claim 10** The method according to claim 9, wherein the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain. **Claim 11** The method according to claim 10, wherein the irradiating induces the light-controllable transcriptional activator to bind to and activate the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both. **Claim 12** The method according to claim 10, wherein said removing induces the light-controllable transcriptional activator to bind to and activate said inducible promoter, thereby causing the expression of said at least one transcription factor, said at least one differentiation factor, or both.

13. The method according to claim 7, wherein said promoter is a constitutive promoter.

14. The method according to claim 13, wherein said at least one light-controllable transcriptional regulatory factor is a light-controllable transcriptional repressor.

15. The method according to claim 14, wherein said light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain.

16. The method according to claim 15, wherein said irradiating induces the light-controllable transcriptional repressor to dissociate from said constitutive promoter, thereby causing the expression of said at least one transcription factor, said at least one differentiation factor, or both.

17. The method according to claim 15, wherein said removing the light induces the light-controllable transcriptional repressor to dissociate from said constitutive promoter, thereby causing the expression of said at least one transcription factor, said at least one differentiation factor, or both.

18. The method according to claim 13, wherein said exogenous nucleic acid further comprises a blocking sequence downstream of said at least one promoter, and said blocking sequence, when present, blocks the expression of at least one first transcription factor, said at least one differentiation factor, or both.

19. The method according to claim 18, wherein said at least one cell further comprises a nucleic acid sequence encoding at least one light-controllable recombinase.

20. The method according to claim 19, wherein said at least one light-controllable recombinase is a photoactivatable recombinase.

21. The method according to claim 20, wherein said photoactivatable recombinase comprises a recombinase or a part thereof fused to a photoactivatable domain.

22. The method according to any one of claims 18 to 21, wherein said blocking sequence is adjacent to a recombinase recognition site recognized by said at least one light-controllable recombinase.

23. The method according to claim 22, wherein said irradiating activates said light-controllable recombinase, thereby resulting in excision of said blocking array and inducing expression of said at least one transcription factor, said at least one differentiation factor, or both.

24. The method according to any one of claims 1 to 23, wherein said at least one cell is a stem cell.

25. The method according to claim 24, wherein said stem cell is a pluripotent stem cell or a multipotent stem cell.

26. The method according to any one of claims 1 to 23, wherein said at least one cell is a fibroblast.

27. The method according to any one of claims 1 to 26, wherein said at least one cell is a human cell, a bovine cell, or a mouse cell.

28. The method according to any one of claims 1 to 27, wherein said desired cell lineage is selected from the group consisting of adipocytes, myocytes, and chondrocytes.

29. The method according to any one of claims 1 to 28, wherein said at least one transcription factor is selected from the group consisting of PPAR gamma, C / EBP alpha, MyoD, MyoG, Myf5, Mrf4, HEYL, KLF4, Pax3, Sox9, Sox5, Sox6, and any combination thereof.

30. The method according to any one of claims 1 to 29, wherein said irradiating further comprises irradiating light to a plurality of cells in said cell population to differentiate each of said plurality of cells into said desired cell lineage, or said removing further comprises removing light from said cell population to differentiate each of said plurality of cells into said desired cell lineage.

31. The method according to any one of claims 1 to 30, wherein said suspension culture has a volume of at least 100 milliliters (mL).

32. The method according to any one of claims 1 to 32, wherein said suspension culture is contained in a bioreactor vessel.

33. The method according to claim 32, wherein said bioreactor vessel has an overall volume of at least 100 milliliters (mL).

34. The method according to any one of claims 1 to 33, wherein said cell population grows on the surface of a microcarrier.

35. The method according to claim 34, wherein said microcarrier is coated with one or more extracellular matrix components.

36. A system for differentiating a cell population, comprising: (a) A cell population in a suspension culture medium, wherein at least one cell of the cell population is engineered to contain an exogenous nucleic acid encoding at least one transcription factor, at least one differentiation factor, or both, which bring about differentiation into a desired cell lineage, and the expression of the at least one transcription factor, the at least one differentiation factor, or both is controlled by light; a cell population; (b) One or more light sources configured to irradiate at least one cell of the cell population with light of a first wavelength; A system comprising the above.

37. The system according to claim 36, wherein the exogenous nucleic acid comprises at least one promoter operably linked to the at least one transcription factor, the at least one differentiation factor, or both.

38. The system according to claim 37, wherein at least one cell is genetically engineered to contain an exogenous nucleic acid encoding at least one light-controllable transcriptional regulatory factor.

39. The system according to claim 38, wherein the promoter is an inducible promoter.

40. The system according to claim 39, wherein the at least one light-controllable transcriptional regulatory factor is a light-controllable transcriptional activator.

41. The system according to claim 40, wherein the light-controllable transcriptional activator comprises a transcriptional activator fused to a light-controllable domain.

42. The system according to claim 41, wherein upon irradiation with light of the first wavelength, the light-controllable transcriptional activator binds to and activates the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both.

43. The system according to claim 41, wherein upon removal of the light of the first wavelength, the light-controllable transcriptional activator binds to and activates the inducible promoter, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both.

44. The system according to claim 38, wherein the promoter is a constitutive promoter.

45. The system according to claim 44, wherein the at least one light-controllable transcriptional regulatory factor is a light-controllable transcriptional repressor.

46. The system according to claim 45, wherein the light-controllable transcriptional repressor comprises a transcriptional repressor fused to a light-controllable domain.

47. The system according to claim 46, wherein the light-controllable transcriptional repressor dissociates from the constitutive promoter upon irradiation with light of the first wavelength, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both.

48. The system according to claim 46, wherein the light-controllable transcriptional repressor dissociates from the constitutive promoter upon removal of the light of the first wavelength, thereby causing the expression of the at least one transcription factor, the at least one differentiation factor, or both.

49. The system according to claim 37, wherein the exogenous nucleic acid further comprises a blocking sequence downstream of the at least one promoter, and the blocking sequence, when present, blocks the expression of the at least one transcription factor, the at least one differentiation factor, or both.

50. The system according to claim 49, wherein the at least one cell of the cell population further comprises a nucleic acid sequence encoding at least one light-controllable recombinase.

51. The system according to claim 50, wherein the at least one light-controllable recombinase is a photoactivatable recombinase.

52. The system according to claim 51, wherein the photoactivatable recombinase comprises a recombinase fused to a photoactivatable domain.

53. The system according to any one of claims 49 to 52, wherein the blocking sequence is adjacent to a recombinase recognition site recognized by the at least one light-controllable recombinase.

54. The system according to claim 53, wherein the blocking sequence is excised by the at least one light-controllable recombinase, thereby inducing the expression of the at least one transcription factor.

55. The system according to any one of claims 36 to 54, wherein the at least one cell is a stem cell.

56. The system according to any one of claims 36 to 55, wherein the stem cell is a pluripotent stem cell or a multipotent stem cell.

57. The system according to any one of claims 36 to 54, wherein the at least one cell is a fibroblast.

58. The system according to any one of claims 36 to 57, wherein the at least one cell is a human cell, a bovine cell, or a mouse cell.

59. The system according to any one of claims 36 to 58, wherein the desired cell lineage is selected from the group consisting of adipocytes, myocytes, and chondrocytes.

60. The system according to any one of claims 36 to 59, wherein the at least one transcription factor is selected from the group consisting of PPAR gamma, C / EBP alpha, MyoD, MyoG, Myf5, Mrf4, HEYL, KLF4, Pax3, Sox9, Sox5, Sox6, and any combination thereof.

61. The system according to any one of claims 36 to 60, further comprising a plurality of microcarriers, wherein the cell population grows on the surface of the plurality of microcarriers.

62. The system according to claim 61, wherein the plurality of microcarriers are coated with one or more extracellular matrix components.

63. The system according to any one of claims 36 to 62, wherein the one or more light sources comprise one or more light emitting diodes (LEDs).

64. The system according to any one of claims 36 to 63, wherein the one or more light sources comprise one or more lasers.

65. The system according to any one of claims 36 to 64, wherein the one or more light sources comprise an incandescent light source.

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